Systems and methods for cryopreservation of biomaterials

ABSTRACT

A cryopreservation system for biological samples is provided. Tire cryopreservation system includes a cooling platform 100 with a 3D printing device that enables a “pick and print” method for processing biological samples 140 for cryopreservation. A syringe or syringes 110 in the 3D printing device picks up biological samples and prints them into a cryogenic environment. A sorting station 200 sorts vitrified samples from unvitrified samples. A warming platform 300 warms the samples using a laser warming system. The cryopreservation system with the sorting station and warming platform are configured for high throughput. Methods for cooling, sorting and warming the biological samples in a high throughput manner are also provided.

This invention was made with government support under OD024430 andEB020537 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Preservation of biological material is valuable in many areas includingfor genetic research, aquaculture development, and biodiversitypreservation. Cryopreservation of germplasm of many important species iscurrently not possible. Examples include the embryos and oocytes ofseveral vertebrate models such as Zebrafish and Xenopus laevis, and manyendangered species (e.g. coral and turtles). Over the past decade, forexample, researchers have increasingly studied and modified the geneticbackbone of the vertebrate model system of the Zebrafish.

The need to preserve germplasm has also become especially urgent inaquatic ecosystems due to coastal pollution, over-fishing, climatechange, and acidifying oceans. Today, about 80% of marine fish stocksare over-exploited, and researchers now list freshwater fish species asone of the most threatened group of vertebrates on the planet. Animportant way to safeguard these unique and endangered species will bethe creation of frozen germplasm banks, which can retain viability foryears (or even centuries) without DNA damage. More specifically, thesebanks offer samples of preserved and protected genetic pools that can beused to ‘seed’ shrinking populations all over the world. Additionally,it allows for easy and inexpensive transport of genetic materials amongliving and/or managed populations. Finally, it vastly improves access tobiomaterials and model organisms for scholarly research.

Maintaining all of these valuable genotypes is expensive, risky, andbeyond the capacity of even the largest stock centers. Moreover, thedifficulty and expense of transporting live colonies makemulti-institutional research rare.

SUMMARY

In a first aspect, the present description relates to a cryopreservationsystem with a cooling platform. The cooling platform includes a syringeholder, the syringe holder including one or more syringes, each syringewith a tip configured to pick and print a biological sample, the syringecoupled to a pressure dispenser wherein the tip of the syringe picks upthe biological sample when the pressure dispenser exerts upward pressurefrom the tip toward the base of the syringe and prints the biologicalsample into a cryogenic environment when the pressure dispenser exertsdownward pressure toward the tip and/or releases the upward pressuretoward the base of the syringe. The syringe can be movably engagedwithin the syringe holder to move from a pick position to a printposition. The biological sample may be printed onto a fibrous wickingmaterial resting on the surface of a highly conductive material. Thehighly conductive material may be resting in cryogenic coolant. Thehighly conductive material may be selected from copper, aluminum,silver, or materials coated with copper, aluminum or silver. The fibrouswicking material may absorb moisture and reduce the moisture absorbed bythe biological sample. The syringe and the tip may be polypropylene. Thebiological sample may be selected from a single cell, multiple cells,aggregates of cells, germplasm, embryos or oocytes. The biologicalsample may be selected from zebrafish embryos, pancreatic islets,Xenopus oocytes, C. Elegans, germplasm, coral germplasm, mammalian,bacteria or protozoa. The biological sample may be at least 0.01 mm indiameter. The biological sample may be a droplet of at least about 0.1μl. The biological sample may be a droplet between about 0.1 μl andabout 40.0 μl. The biological sample can include laser absorbers and/orcryoprotective agents. The cooling platform may be a high throughputsystem including two or more syringes with tips for picking and printingmultiple biological samples. The system may further include a sortingstation. The sorting station can sort the biological samples andseparate vitrified biological samples from crystallized biologicalsamples, wherein the sorting station may include a microfluidics basedsorting device with channels sized for flow of the samples in a fluid, alight source and a detector. The sorting may be performed prior to thefreezing of the biological sample. The sorting may be performed when thebiological sample is at a cryogenic temperature. The system may includea warming platform. The warming platform can include a cryoscoop forremoving the biological sample from a cryogenic environment. The warmingplatform can further include a laser for warming the biological sampleat a cryogenic temperature to a desired temperature. The laser may warmthe biological sample in 1-30 milliseconds to room temperature. Thecryoscoop may be reusable.

In another aspect, the present description relates to a sorting station.The sorting station can sort biological samples and separate vitrifiedbiological samples from unvitrified biological samples. The sortingstation can include a sorting device with channels sized for flow ofbiological samples in a fluid, a light source and a detector. Thesorting station may also include a pressurized air tank operablyconnected to the detector and a buffer reservoir wherein the detectorcan detect a vitrified sample from a unvitrified sample and thepressurized air tank operably connected to send a pulse of pressurizedair to the buffer reservoir through an airline resulting in a pulse ofbuffer fluid entering the channel in the sorting station to alter thepathway of the biological sample in the channel closest to the distalend of the pulse of the buffer fluid. The sorting station may separatethe vitrified samples and the unvitrified samples into differentchannels. The sorting station can be a microfluidics based sortingstation. The sorting may be performed prior to the freezing of thebiological sample. The sorting may be performed when the biologicalsample is at a cryogenic temperature.

In a further aspect, the present description can include a warmingplatform. The warming platform can include a cryoscoop for removing thebiological sample from a cryogenic environment. The warming platform canfurther include a laser for warming the biological sample at a cryogenictemperature to a desired temperature. The laser may warm the biologicalsample in 1-30 millisecond to room temperature. The cryoscoop may bereusable.

In another further aspect, the present description relates to a methodfor cryopreservation of a biological sample. The method can includepicking up the biological sample with a syringe having a tip, whereinthe syringe is engaged in a syringe holder of a cooling platform, thesyringe coupled to a pressure dispenser wherein the tip picks up thebiological sample when the pressure dispenser exerts upward pressurefrom the tip toward the base of the syringe. In other words, thebiological sample associates with the tip due to the upward pressurewithin the syringe and tip. The method also includes printing thebiological sample wherein the sample is printed when the pressuredispenser exerts downward pressure toward the tip and/or releases theupward pressure toward the base of the syringe. The biological samplecan be printed into a cryogenic environment. The cooling platform may bea high-throughput system including one or more syringes. The method mayinclude cryopreserving about 20-400 biological samples in about 1minute. The cooling rate may be about 1000 to about 10,000 ° C./min. Thebiological sample can be printed onto a fibrous wicking material restingon the surface of a highly conductive material and wherein the highlyconductive material and the fibrous wicking material are at a cryogenictemperature. The highly conductive material may be resting in cryogeniccoolant. The highly conductive material may be selected from copper,aluminum, silver or materials coated with copper, aluminum or silver.The fibrous wicking material may absorb moisture and reduce the moistureabsorbed by the biological sample. The syringe and the tip may bepolypropylene. The biological sample may be selected from a single cell,multiple cells, aggregates of cells, germplasm, embryos or oocytes. Thebiological sample may be selected from zebrafish embryos, pancreaticislets, Xenopus oocytes, C. Elegans, germplasm, coral germplasm,mammalian, bacteria or protozoa. The biological sample may be at least0.01 mm in diameter. The biological sample may be a droplet of at leastabout 0.1 μl. The biological sample may be a droplet between about 0.1μl and about 40.0 μl. The biological sample may include laser absorbersand/or cryoprotective agents. The cooling platform may be a highthroughput system including two or more syringes with tips for pickingand printing multiple biological samples. The method may includeapplying pressure when vitrified or crystallized samples are detected bya detector to separate the vitrified biological samples from thecrystallized biological samples.

The method can include sorting the biological samples by sorting andseparating vitrified biological samples from crystallized biologicalsamples. The method can include sorting with a microfluidic sortingsystem. The sorting station can be a microfluidics based sorting stationand may include channels for passage of the samples in a fluid, a lightsource and a detector. The method can include sorting prior to coolingthe biological sample to a cryogenic temperature. The method can includesorting after freezing the biological sample to a cryogenic temperature.

The method can include warming the cryopreserved biological sample witha warming platform. The warming method can include incubation of thebiological sample at room temperature. The warming method can includeremoving the biological sample from a cryogenic environment by acryoscoop. The warming method can include laser assisted warming. Thelaser assisted warming method may warm the biological sample in about1-30 milliseconds from a cryogenic temperature to room temperature. Thewarming rate may be about 400,000-24 million ° C./min. The warmingplatform may be a high-throughput system including multiple cryoscoops.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a simplified block diagram of a cooling platform of acryopreservation system.

FIG. 1B is a schematic diagram of one exemplary embodiment of a pick andprint device printing an embryo to a cryogenic copper dish.

FIG. 1C is a schematic diagram of a top view of a high-throughputsyringe holder.

FIG. 2A is a simplified block diagram of a sorting station of acryopreservation system.

FIG. 2B is one exemplary embodiment of a schematic diagram ofmicrofluidic optical sorting device for vitrified and crystallizedembryos.

FIG. 3A is a simplified block diagram of a warming platform of acryopreservation system.

FIG. 3B is a schematic diagram of a laser-warming device of a samplewith a cryoscoop.

FIG. 3C is a schematic diagram of convective warming for vitrified CPAdroplet.

FIG. 3D is a schematic diagram of laser warming method for vitrified CPAdroplet.

FIG. 3E shows photographs of the undesirable crystallization that occursin convective warming, but not in laser warming.

FIG. 4A is a schematic diagram of components of a “pick and print”cryopreservation system showing a droplet printed directly to liquidnitrogen which leads to vitrification failure. A photograph (middle) andan X-ray diffraction (XRD) image (right) of the crystallization of theCPA droplet (1.2 mm diameter, 2 M propylene glycol+1 M trehalose±3 ODGNP) when printed directly into liquid nitrogen are also shown.

FIG. 4B is a schematic diagram components of a “pick and print”cryopreservation system showing a droplet printed onto cryogenic copperdish which leads to vitrification success. A photograph (middle) and anXRD image (right) of the vitrification of the same CPA droplet as inFIG. 4A but when printed onto cryogenic copper dish are also shown.

FIGS. 4C, 4D and 4E are photographs of vitrified droplets withoutislets, vitrified droplet with islets and non-vitrified droplets withislets, respectively.

FIG. 5A is a photograph of the cryopreservation system of the embryo“pick & print” system. This embodiment includes a custom-built 3Dprinter, a pressure dispenser, and a high precision tip.

FIG. 5B is an optical image of a printed droplet falling from the tip.

FIG. 6A is a photograph of microfluidic device with fitted tubing.

FIG. 6B is a photograph of droplets flowing in mineral oil in a 200 μmwide microfluidic channel Dark frozen droplets are circled compared tolighter unfrozen droplets.

FIG. 7 is a flowchart of one embodiment of a method of cryopreservationof a biological sample described herein.

FIG. 8 is a plot of the laser fluence rate obtained for the 1064 nmlaser by varying input voltages from 155V to 400V (10V increments) andpulse width from 0.5 ms to 20 ms (0.5 ms increments to 5 ms, and 1 msincrements till 20 ms).

FIG. 9 is a plot showing the minimum laser fluence rate required to melta single 1 microliter droplet containing 2M propylene glycol and 1MTrehalose but varying concentrations GNR. It shows that amount of laserpower decreases with increasing gold concentration.

FIGS. 10A-10C are schematic diagrams of a high throughput laser warmingsetup.

FIGS. 11A-11C are plots of a simulation of the temperature distributionin a droplet at different GNR concentrations.

FIG. 12A is a plot of the effect of laser energy and droplet volume oncell viability.

FIG. 12B is a plot of the effect of laser energy and the concentrationof the gold nanorods (GNR) concentration on cell viability.

FIG. 12C is a bar graph of the cell viability in samples after use ofdifferent warming protocols.

FIGS. 13A-D are schematic drawings of a perspective, side view, top viewand bottom view, respectively, of a cryoscoop.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present description is directed to systems and methods forcryopreservation of biological materials and warming of biologicalmaterials. The cryopreservation system includes a cooling platform. Thecooling platform can include a 3D printing device. The 3D printingdevice can enable a “pick and print” method for processing biologicalsamples by picking/lifting a biological sample from its environment,e.g. at room temperature, and printing the sample into a cryogenicenvironment. The cryopreservation system may also include a microfluidicsorting station and/or a warming platform. In one embodiment, thecryopreservation system is a high-throughput system and includes ahigh-throughput cooling platform, a high-throughput sorting stationand/or a high throughput warming platform.

High-throughput cryopreservation of biological material, for example,cells and aggregates (i.e. pancreatic islets), embryos or oocytes (i.e.other vertebrate biomedical models) and commercially relevant orendangered species (i.e. agriculture, aquaculture and biodiversity) canbe performed using the systems and methods described herein.Cryopreservation of germplasm of aquatic species is increasingly vitalfor biomedical research, aquaculture and maintenance of biodiversity. Insome exemplary embodiments, biological material that can becryopreserved can include, for example, embryos and oocytes of fish andamphibians. Well-established, reproducible cryopreservation ofbiological material can provide a unique opportunity to preserve andexpand the use of important biological material.

Although methods are known for cryopreservation of small biologicalsamples, i.e. less than about 0.1 μl droplets, cryopreservation oflarger samples has been challenging. Problems related to crystallizationof the droplets can adversely affect the sample and/or destroy thesample. Crystallization of the biological sample during cooling and/orwarming can lead to disruption of the cellular membranes and otherstructures that can destroy the integrity of the sample. Similarly, whenwarming cryopreserved biological samples, i.e. greater than 0.1 μl,uneven warming can lead to destruction in the integrity of the sampleand lower sample survival rates.

Cryopreservation can allow viable cells and tissues to be preserved overtime in the hypothermic, frozen, or vitrified (glassy) state. Thisdisclosure describes systems, compositions and methods that may be usedto cool biological samples and warm cryopreserved biological samplesfrom cryogenic temperatures. The systems, methods and compositionsdescribed herein are useful in, for example, cooling millimeter-sizedcryopreserved biological samples such as, for example, zebrafishembryos, marine germplasm, and/or other 10 micrometer tomillimeter-sized model systems such as, for example, mammalian cells,pancreatic islet cells, stem cells, biopsies of tissues, etc. (0.1 μl to40 μl droplets).

The cryopreservation systems described herein advantageously can be usedin methods to process biological samples for long-tem storage bycryopreservation and also rewarming of the cryopreserved material.High-throughput techniques can be adapted for processing a large numberof samples during cryopreservation, sorting and warming. Thecryopreservation systems can use optical and/or laser technology forsorting, manipulation, analysis and warming of cryopreserved tissues.Biological samples that are greater than about 0.1 microliter dropletsor greater than about 1 micrometer (1 μm) can advantageously becryopreserved using the methods described herein. Biological samplesless than about 0.1 microliter of droplets or less than about 1micrometer may also be cryopreserved using the methods described herein.

The systems described herein can be used to attain the critical coolingrates (CCR) and critical warming rates (CWR) needed for physical (nocrystallization) and biological (no toxicity) cryopreservation successin larger biological samples. The systems and methods described hereincan preserve and restore the integrity of the biological samples uponwarming. The cooling of the biological sample can result invitrification of the sample. In one embodiment, this description isdirected to systems and methods that can include cooling and warmingplatforms that can achieve sufficiently high CCRs and CWRs to producelive zebrafish embryos (800 μm) post-cryopreservation.

DEFINITIONS

Various terms are defined herein. The definitions provided below areinclusive and not limiting, and the terms as used herein have a scopeincluding at least the definitions provided below.

The terms “preferred” and “preferably”, “example” and “exemplary” referto embodiments that may afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred orexemplary, under the same or other circumstances. Furthermore, therecitation of one or more preferred or exemplary embodiments does notimply that other embodiments are not useful, and is not intended toexclude other embodiments from the inventive scope of the presentdisclosure.

The singular forms of the terms “a”, “an”, and “the” as used hereininclude plural references unless the context clearly dictates otherwise.For example, the term “a tip” includes a plurality of tips.

Reference to “a” chemical compound refers one or more molecules of thechemical compound, rather than being limited to a single molecule of thechemical compound. Furthermore, the one or more molecules may or may notbe identical, so long as they fall under the category of the chemicalcompound.

The terms “at least one” and “one or more of” an element are usedinterchangeably, and have the same meaning that includes a singleelement and a plurality of the elements, and may also be represented bythe suffix “(s)” at the end of the element.

The terms “about” and “substantially” are used herein with respect tomeasurable values and ranges due to expected variations known to thoseskilled in the art (e.g., limitations and variability in measurements).

The terms “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The terms “comprises,” “comprising,” and variations thereof are to beconstrued as open ended—i.e., additional elements or steps are optionaland may or may not be present.

Unless otherwise specified, temperatures referred to herein are based onatmospheric pressure (i.e. one atmosphere).

“Cryopreservation” as referred to herein relates to preservation of abiological sample at cryogenic temperatures. Cryopreservation includescooling/freezing the biological sample below subzero temperatures inorder to shut down metabolic/chemical activity which can provide longterm storage of biomaterials. Cryopreservation of a biological samplemay also include warming the biological sample to recover the functionactivity of the biological sample.

“Cryogenic” or “Cryogenic temperature” as referred to herein relates toa temperature below sub-zero. Cryogenic temperature can be from −80° C.(112° F.) to absolute zero (−273° C. or −460° F.).

“Cryogenic coolant” as referred to herein relates to a substance that isat a cryogenic temperature, e.g. liquid nitrogen, slush nitrogen.

“Pick” and/or “picking” as referred to herein relates to association ofa biological sample with a syringe tip when pressure is exerted upwardfrom the tip through the syringe toward the base of the syringe. Theupward pressure enables the sample to be associated and remainassociated with the syringe tip as the syringe moves, for example, froma pick position to a print position. The sample may be associated bybeing completely within a syringe and/or the syringe tip, partiallyoutside of the syringe tip, substantially outside of the syringe tip orcompletely outside of the syringe tip.

“Print” and/or “Printing” as referred to herein relates to releasing ordepositing a biological sample. The sample, for example, can bedeposited or released into a cryogenic environment. The releasing ordepositing of the biological sample can occur when the upward pressurefrom the tip to the base of the syringe is released or if there isdownward pressure applied through the syringe from the base of thesyringe toward the tip of the syringe.

“Cryoscoop” as referred to herein relates to a cryoresistant tool thatcan handle a biological sample. The cryoscoop can, for example, remove asample from a cryogenic environment. The biological sample may also restor reside in the cryoscoop during a warming protocol.

“Vitrification” as referred to herein relates to a biological samplethat has attained a glassy, amorphous structure when cryopreserved.Vitrified samples have less 0.1% V/V of ice crystallization in thesample.

“Crystallized” sample as referred to herein relates to a biologicalsample that has attained some crystalline structure and may not producea viable biological sample upon warming to room or physiologicaltemperature. Crystallized samples may also be referred to herein asunvitrified samples or non-vitrified samples. These terms are usedinterchangeably herein.

“Sorting” as referred to herein relates to identifying vitrified samplesfrom unvitrified samples and separating the vitrified samples from theunvitrified samples. The separation can be performed, for example, byaltering the path of the samples such that vitrified samples aredirected to a different destination, e.g. channel, than unvitrifiedsamples.

“High-throughput” as referred to herein relates to the use of automationof a system to rapidly process a large number of samples in short amountof time.

“Germplasm” as referred to herein relates to living genetic resourcesthat are maintained for the purpose of animal and plant breeding,preservation and other uses.

“Biological specimens” or “biological samples” are used interchangeablyand as referred to herein relate to cells, germplasm, cell aggregates(i.e. pancreatic islets), embryos, oocytes and the like. The germplasmcan be from a variety of species including, for example, coralgermplasm, mammalian germplasm and the like. The biological samples canbe unicellular organisms such as bacteria, protozoa and the like. Theembryos and oocytes can be, for example, from fish, amphibians, mammals,humans and other vertebrates. The biological samples can be related tocommercially relevant or endangered species (i.e. agriculture,aquaculture and biodiversity).

Biological samples can include other components to aid in thecryopreservation process, e.g. cryopreserving agent, laser absorberssuch as gold nanorods and the like and/or buffer or other media that arepresent when the biological sample is prepared, transferred and/orcryopreserved. The size of the biological sample may be characterized byvolume as a droplet. The droplet, for example, includes the biologicalsample. The droplet may further include cryoprotective agent(s), laserabsorbers, a buffer or media and/or other agents to aid in thecryopreservation. The size of the biological sample may be characterizedby the diameter of the biological sample or specimen and/or the volumeof the droplet.

In the following detailed description of illustrative examples,reference is made to specific embodiments by way of drawings andillustrations. These examples are described in sufficient detail toenable those skilled in the art to practice what is described, and serveto illustrate how elements of these examples may be applied to variouspurposes or embodiments. Other embodiments exist, and logical,mechanical, electrical, and other changes may be made.

Features or limitations of various embodiments described herein, howeverimportant to the examples in which they are incorporated, do not limitother embodiments, and any reference to the elements, operation, andapplication of the examples serve only to define these illustrativeexamples. Features or elements shown in various examples describedherein can be combined in ways other than shown in the examples, and anysuch combinations is explicitly contemplated to be within the scope ofthe examples presented here. The following detailed description doesnot, therefore, limit the scope of what is claimed.

All patents, publications or other documents mentioned herein areincorporated by reference.

In one embodiment, the present description includes a cryopreservationsystem with a cooling platform for cryopreservation of a biologicalsample. FIG. 1A is a simplified block diagram of an exemplary embodimentof cooling platform 100. FIG. 1B is a schematic diagram of one exemplaryembodiment of a “pick and print” 3D print device for cooling platform100. Cooling platform 100 includes 3D print device 104 that includessyringe 110 with tip 120 engaged or movably held within syringe holder106. In one embodiment, tip 120 is a high precision tip. Coolingplatform 100 can include pressure dispenser 130 that can exert pressurewithin and/or through syringe 110. Pressure dispenser 130 can beoperably connected to exert either upward pressure, downward pressure orno pressure on syringe 110. Pressure dispenser 130 may exert pressuredownward from base of syringe 110 towards tip 120 as shown, for example,in FIG. 1B when printing sample 140. Pressure dispenser 130 may exertpressure upward from tip 120 to the base of syringe 110 as shown, forexample, in FIG. 1B when picking up sample 140. Printing may also occurwhen the upward pressure is released by pressure dispenser 130 and thereis no longer sufficient pressure upward to associate sample 140 to tip120.

Syringe 110 can be used to pick up sample 140 resting in media 146. Inone exemplary embodiment, sample 140 can be an embryo and media 146 canbe embryo media. Tip 120 can pick up droplet 144. Droplet 144 caninclude sample 140 and surrounding media 146. In one embodiment of apickup setup, syringe 110 and tip 120 are purchased from Nordson EFD,East Providence, R.I. Syringe 110 can be made from a polypropylene blend(3cc, 47012074, EFD). Tip 120 attached to the syringe can be made ofstainless steel, for example, with an inner diameter of 0.41 mm(#7018281, EFD). Tip 120 made from other materials such as polyethylene,nylon, or glass with various diameters can also be employed and maybeattached, for example, to syringe 110 through luer lock threaded hubsfor the pick-up process. Sample 140 can include biological material andagents to aid in the cryopreservation process such as cryopreservationagents, laser absorbers as described in further detail below.

Pressure dispenser 130 is calibrated to exert upward pressure from tip120 through syringe 110 toward the base of syringe 110 when tip 120contacts sample 140 in media 146 to pick up sample 140. The upwardpressure provided by pressure dispenser 130 at tip 120 and the surfacetension between media 146 and tip 120 can enable syringe 110 to picksample 140 in droplet 144 from media 146 by providing sufficientpressure for sample 140 to maintain association to tip 120 as shown, forexample in FIG. 1B. Syringe 140 can slide or move from media 146 towardcryovessel 154 in order to print sample 140 into a cryogenicenvironment. Sample 140 associated with tip 120 can be then printed in adesired manner. Printing of sample 140 can include changing thedirection of the pressure from pressure dispenser 130 to exert downwardpressure toward tip 120 that can release sample 140. Printing of sample140 can also include release of the pressure from pressure dispenser130. The lack of upward pressure can release sample 140 from tip 120.

In one embodiment of a high-throughput 3D printing device, syringeholder 106 can include multiple stations or positions, notated as I, II,III and so on in FIG. 1C, to engage multiple syringe(s) 110. FIG. 1C isa top view of one exemplary embodiment of syringe holder 106 in ahigh-throughput embodiment of cooling platform 100. Syringe holder 106can include multiple positions, I, II, III, IV, and so on, forpositioning multiple syringes 110 at each of the positions. Thesemultiple syringes can be configured to “pick” sequentially and/orsimultaneously. Syringes can be continuously circulating through each ofthe positions in a continuous manner. In one embodiment, syringe(s) 110are positioned at each of the positions. Pressure dispenser 130 andsyringe(s) 110 in syringe holder 106 are configured in a manner that afirst syringe at position I can pick up a sample and is conveyed toposition II after sample pickup. Syringes can sequentially and/orcontinuously move through each of the positions in syringe holder 106.Another position, for example, position III, can be configured to be theprinting position. When, for example, a first syringe 110 enters theprinting position, e.g. position III, the first syringe 110 can printsample 140. After printing sample 140, the first syringe 110 at positionIII can be conveyed to the next position and continue to cycle throughthe positions in syringe holder 106 until the first syringe arrives backat position I to pick up another sample 140. In one embodiment, all ofthe positions include syringe(s) 110 and as a first syringe moves to asecond position, a second subsequent syringe can be conveyed to positionI to pick up another sample and so on. At any given time, a syringe atposition I can pick up a sample and a syringe at position III can printa sample and syringes at other positions are conveyed through syringeholder 106 until they arrive at the pick or print positions to carry outeither the pick task or the print task. FIG. 1B exemplifies one “pick”syringe and one “print” syringe.

In one embodiment, a first syringe 110 can be at position I placed oversamples 140. When pressure dispenser exerts upward pressure in syringe110 at position 1, sample 140 associates with tip 110. After associationwith tip 110, first syringe 110 can move to position II with associatedsample 140. When the first syringe moves to position II whilemaintaining the upward pressure from the pressure dispenser, a secondsyringe may move to position I in order to pick up another sample 140.The first syringe 110 may be further conveyed to position III whilemaintaining the upward pressure and the second syringe may be conveyedto position II while a third syringe enters position I. Syringe 110 atposition III can have access to a cryogenic environment. In oneembodiment, in position III, syringe 110 and tip 120 associated withsample 140 can be over cryovessel 154 with coolant 156 as shown in FIG.1B in order to print sample 140 into a cryogenic environment. Sample 140can be released in position III into a cryogenic environment uponexertion of downward pressure from the pressure dispenser throughsyringe 110 at position III. The specific position in the syringe holderwhere the samples are picked up by a syringe and the specific positionin the syringe holder where the samples are printed into a cryogenicenvironment can vary. The number of positions between the “pick” aspectand the “print” aspect of the 3D printing device can vary and all arewithin the scope of this description. In a high-throughput system, acooling platform can include two or more syringes, five or moresyringes, 10 or more syringes and all are within the scope of thisdescription.

In the embodiments of simultaneously picking up, multiple syringes canbe configured to “pick” simultaneously and conveyed to multiple printpositions. All syringes 110, in position I, II, III and so on, insyringe holder 106 can be placed over samples 140 at the same time. Whenpressure dispenser exerts upward pressure in syringes 110 at all thepositions, many samples 140 associate with tips 110. Then syringes 110can be moved to a printing position while maintaining the upwardpressure. Samples 140 can be released at the printing position into acryogenic environment upon exertion of downward pressure from thepressure dispenser through all syringes 110 in syringe holder 106.Hence, multiple syringes can “print” simultaneously for a furtherhigh-throughput system.

Many variations in the size and configuration of the syringe holder,number of syringes, location of the pick and print positions arepossible and all are within the scope of this description. Embodimentswith multiple pick positions and multiple print positions within asyringe holder are also within the scope of this description. Samplescan be picked simultaneously or sequentially. Samples can be printedsimultaneously or sequentially.

Sample 140 can be printed into or onto a variety of cryogenicenvironments. Sample 140 may be printed into a cryogenic environmentsuch as vessel 154 that includes cryogenic coolant 156. Cryogeniccoolant 156 can include, for example, liquid nitrogen. Cryogenic coolant156 may also include slush nitrogen. Other cryogenic coolants such asethanol, methanol, FC 770 oil (3M) may also be used and all are withinthe scope of this description. In one embodiment, sample 140 can beprinted onto cryogenic surface 150 that is at a cryogenic temperature.The cooling of sample 140 can occur to achieve critical cooling rates(CCR) as further described herein.

In one embodiment, cryogenic surface 150 can be a highly conductivematerial. The highly conductive materials are generally cryoresistant,e.g. maintain integrity at cryogenic temperatures, in order to receive abiological sample. Highly conductive materials that can be used as acryogenic surface include, for example, copper, silver, aluminum, andthe like. The cryogenic surface can also include other materials thatare coated with copper, silver, aluminum, and the like. Althoughcryogenic surface 150 is exemplified in FIG. 1B as a dish, other shapesmay also be used as a cryogenic surface. Cryogenic surface 150 may alsoinclude a flat surface, e.g. without any sidewalls. Cryogenic surface150 can include a variety of thicknesses and the thickness is such thatthe surface receiving sample 140 can be maintained at a cryogenictemperature.

In some embodiments, the highly conductive material may be overlaid by afibrous wicking material that is also at the cryogenic temperature andsample 140 is printed onto the fibrous wicking material overlaid on thehighly conductive material. The fibrous wicking material may be placedor be resting on the highly conductive material to advantageously wickany moisture that may be present in the sample. The fibrous wickingmaterial can be, for example, a fibrous tissue. The thickness of thefibrous wicking material can vary and is within the thickness such thatthe surface receiving the biological sample can be maintained at acryogenic temperature. The fibrous wicking material can have a thicknessof at least about 0.1 mm. In some embodiments, the thickness of thefibrous wicking material is between about 0.1 mm and about 2 mm.Thickness outside of this range are also within the scope of thisdisclosure. The highly conductive material may or may not have thefibrous wicking material when a sample is placed in the cryogenicenvironment.

Sample 140 may be maintained at a cryogenic temperature on cryogenicsurface 150. Alternatively, sample 140 may be allowed to enter thecryogenic coolant 156. In one embodiment, sample 140 is printed ontocryogenic surface 150 overlaid with a fibrous wicking material. Printedsample 140 can be allowed to drop into coolant 156 by tilting cryogenicsurface 150 in order for sample 140 to roll or drop into coolant 156.

Cooling platform 100 can include other components to carry out thefunctions of the pick and print device as indicated in a block diagramin an exemplary embodiment shown in FIG. 1A. Syringe 110 with tip 120are operably engaged in syringe holder 106. Pressure dispenser 130 isoperably connected to syringe holder 106 to exert upward or downwardpressure to syringe(s) 110 at the desired positions. Operation of system100 can include input/output (I/O) circuitry 124 to allow, for example,the timing, the amount of pressure, the direction of pressure to beapplied by dispenser 130 through syringe 110 and tip 120. Computingsystem 128, for example, a microprocessor, can be configured for input134 a and/or output 134 b in order to execute the cryopreservationprocess. Input 134 a can include, for example, information entered by anend user or a button to initiate the cryopreservation process. Computingsystem 128 can be coupled through I/O circuitry 124 to control pressuredispenser 130 and/or syringe holder 106. Computing system 128 canactivate pressure dispenser 130 to control the direction of pressure,the amount of pressure in a syringe and the timing of pressure appliedthrough a syringe. The direction of the pressure may be upward from thetip of the syringe towards the base of syringe, for example, whenpicking up a sample. The direction of the pressure may be downward fromthe base of the syringe toward the tip of the syringe, for example, whenprinting a sample. In some embodiments, the pressure dispenser mayrelease the upward pressure after picking up a sample to print thesample. In other words, printing may occur when the pressure dispenserapplies little downward pressure or no pressure. I/O circuitry 124 caninclude, for example, digital-to analog converters, analog-to-digitalconverters, switchable outputs, etc. Power supply 116 is provided andcan be, for example, a portable power sources such as a battery, anon-portable power source or the like. The power supply may be arechargeable either through connection to another electrical powersource, a solar cell or the like.

Input/output circuitry 124 is also illustrated coupled to computingsystem 128. Computing system 128 can be a microprocessor. This mayinclude any type of input or output device including a display, keyboardor manual input, audible output, digital output such as a USB orEthernet connection, an RF (radio frequency) or IR (infrared) inputand/or output, a cellular data connection, an Ethernet connection, etc.Example RF connections include but are not limited to BLUETOOTH®connections or other short distance communication techniques, WIFIconnections, or others. Cellular phone connections allow the device tocommunicate using a cellular phone network for communicating data and/orproviding optional voice communication.

In some embodiments, the cryopreservation system may also include asorting station. The sorting station can include a sorting device thatcan be used to sort viable and/or vitrified biological samples fromnon-viable, unvitrified and/or crystallized biological samples. Sortingmay be conducted prior to cryopreservation, after cryopreservationand/or proceeding the warming of the cryopreserved sample. Sortingstation may be a standalone device. Alternatively, it may be coupled orintegrated to a cooling platform and/or a warming platform. In oneembodiment, the sorting station is coupled to the cooling platform suchthat samples that have been cooled to a cryogenic temperature areconveyed to the sorting station to identify and separate the vitrifiedsamples from crystallized or partially crystallized samples.

In some embodiments, the viability of the samples can be determined toeliminate non-viable, unvitrified biological samples. Prior to freezing,viable, vitrified biological samples may be selected using any suitablemethod including, for example, manual selection, centrifugation, or flowcytometry.

In one embodiment, the sorting device can include a microfluidic systemthat could allow for high throughput optical sorting of viable versusnon-viable specimen at room temperature prior to printing into liquidnitrogen. Sorting of biological samples prior to freezing has beendescribed, for example, in WO2017/184721 A1 by Bischof et al. andincorporated herein by reference. The selection of viable samples may beautomated in a high-throughput system.

Printing of biological samples into a cryogenic environment can resultin a mixture of vitrified and unvitrified or crystallized samples.Vitrified samples are samples that have become solid without freezingand these samples are non-crystalline amorphous solids. Crystallizedsamples have attained at least some crystalline characteristics and arenot conducive to regaining all or substantial amount of biologicalactivity upon warming. A sorting station may be used to separate thevitrified samples from the crystallized or unvitrified samples in orderto eliminate the crystallized samples from long-term storage and/orwarming. In one embodiment, the crystallized samples may be discarded orseparated from the vitrified samples. In one embodiment, the biologicalsamples may be sorted to separate the vitrified and crystallized samplesafter the biological sample is at a cryogenic temperature. Biologicalsamples described herein may be sorted using a sorting station after thebiological samples have been cryopreserved or printed into a cryogenicenvironment. The biological samples may be sorted using a flow systemwhere the samples flow in a fluid in a channel and each sample isevaluated by a detection system to determine if a sample is vitrified orunvitrified. In one exemplary embodiment, the samples may be sortedbased on an optical detection system.

In one exemplary embodiment shown in FIG. 2A and FIG. 2B, a microfluidicoptical platform can be used as the sorting device in the sortingstation. Samples 240 a and 240 b are shown as vitrified and crystallizedsamples, respectively. In FIG. 2A and FIG. 2B, sorting station 200includes sorting device 204 with channel 262, fluid 266 in channel 262,light source 270, detector 272, and pressure source 274. Channel 262 canhave fluid 266 flowing in the direction indicated and have a thicknessto accommodate samples 240 a and 240 b. Channel 262 can also include aforked junction to generate two separate channels 262 a and 262 b.Channels 262, 262 a and 262 b are sized to allow flow of samples 240 aand 240 b in fluid 266. Light source 270 and detector 272 are placedalong the flow path of samples 240 a and 240 b in fluid 266 Channelwidth may be narrowed at detection to accelerate the flow and spacesamples further apart during detection. As samples 240 a and 240 b passacross or past the path of light source 270, detector 272 can ascertainvitrified sample 240 a from crystallized sample 240 b based on theinteraction of light source 270 with samples 240. In one embodiment,light source 270 interacts differently with sample 240 a than sample 240b and this difference is detectable by detector 272. Detector 272 isoperably connected to pressure source 274. Single or multiple (encodedarrays) detector output signals may be used. When detector 270 detectsunvitrified sample 240 b, signal 272 a can be sent to CPU 276. CPU 276generates signal 276 a to actuate pressure source 274. Signal 272 a and276 a can be received and/or transmitted through a wired or wirelessconnection. Pressure source 274 can calibrate the pressure based on theoutput of detector 272 to direct vitrified samples 240 a into channel262 a and crystallized samples 240 b into channel 262 b as exemplifiedin FIG. 2B. In one embodiment, pressure source 274 can include apressure regulator/solenoid valve. Airline 274 a connects pressuresource 274 to buffer tank 280 including buffer 284. A brief flow of airwith each pressure pulse when detector 272 detects unvitrified sample240 b can be injected into buffer tank 280. Pressurized air from airline274 a in response to a pressure pulse can enter buffer tank 280 andresult in pressure pulsing buffer 284 through buffer line 280 a intochannel 262 c. A pulse of buffer fluid 284 into channel 262 c can leadto sorting unvitrified embryos 240 b by deflection into channel 262 b.Alternatively, detector 272 may detect vitrified embryos 262 b and sendsignal 272 a upon detection of vitrified samples 262 b. In suchembodiments, buffer 284 in buffer tank 280 may be pulsed into channel262 c when vitrified samples are detected. Alternate methods ofseparating vitrified and unvitirified samples may be used and all arewithin the scope of this description.

In embodiments of cryopreserved biological samples, channels 262, 262 a,262 b and 262 c include materials that are compatible with cryogenictemperatures. Fluid 266 within channels 262, 262 a and 262 b can becompatible with the cryogenic temperatures to maintain samples 240 a and240 b at a cryogenic temperature and enable samples 240 a and 240 b toflow through channels 262, 262 a and 262 b. Fluids can be, for example,FC770 oil (3M) and the like.

In the schematic diagram of an exemplary embodiment shown in FIG. 2B,light source 270 can be, for example, visible light or near infraredthat is focused at a position where samples 240 a and 240 b flow throughin channel 262. As samples 240 a and 240 b pass across the path of lightsource 270, detector 272 such as a light sensor or an infrared sensordetects a reading and is able to discern if a sample is vitrified orcrystallized based on the differing results for sample 240 a versussample 240 b. An optional user input 234 is provided. For example, thisinput can be a single button allowing an operator to initialize a test,or can be a more complex input such as a numerical keypad or of anumeric keypad allowing an operator to update parameters such asthreshold values used by station 200. In the simplified block diagram ofFIG. 2A, a computer identified as computing system 228 is used toperform the sorting. Computing system 228 can be coupled to light source270 and detector 272 through I/O circuitry 224. I/O circuitry 224 caninclude, for example, digital-to-analog converters, analog-to-digitalconverters, switchable outputs, etc. Single or multiple (encoded arrays)detector output signals may be used.

Although any appropriate components may be employed, in one embodiment,the source 270 comprises a laser, for example a 532 nm green laser (i.e.LRS-0532-PFM_00200-03, LaserGlow Technologies Inc.). Focusing lightsource 270 can comprise for example a plano-convex focusing lens. Asuitable infrared detector includes an infrared camera (A20 or E30, FLIRInc) or infrared detector (MLX90614, Melexis). However, the presentdescription is not limited to this configuration.

FIG. 6 is a photograph of microfluidic device with fitted tubing (FIG.6A), and dark frozen (circled) and light unfrozen liquid dropletsflowing in mineral oil (FIG. 6B). The microfluidic device with thefitted tubing is from Metcalf, Boyer, and Dutcher, “Interfacial Tensionsof Aged Organic Aerosol Particle Mimics Using a Biphasic MicrofluidicPlatform”, Environmental Science and Technology, 50, 1251-1259, 2016,incorporated herein by reference. FIG. 6B illustrates that an opticaldetection system can be used to identify and separate vitrified andunvitrified embryos based on opacity.

A variety of appropriate components and configurations may be used asdescribed, for example, in Xi, Heng-Dong et al. “Active droplet sortingin microfluidics: a review. Lab on a Chip, Issue 5, 2017, incorporatedherein by reference.

The present description also includes a cryopreservation system with awarming platform. The warming platform can be used to warm thecryopreserved biological sample to room temperature or otherphysiological temperatures. In other words, the warming platform is usedto warm the cryopreserved sample from a cryogenic temperature to anon-cryogenic temperature. The warming platform can be a stand-alonesystem. Alternatively, the warming platform may be coupled or integratedto a cooling platform and/or a sorting station.

In one exemplary embodiment, warming platform 300 as shown in FIGS. 3Aand 3B. In schematic diagram of FIG. 3B, warming device 304 can be usedto warm cryopreserved sample 340. Sample 340 can be resting, forexample, in cryogenic coolant 356 in vessel 354. Cryopreserved sample340 can include laser absorbers such as GNRs. Cryoscoop 384 may be usedto remove the cryopreserved biological sample. Cryoscoop 384 can includehandle 388, and may be modified at the scooping end, for example, as aspoon or ladle, by the addition of scoop 392 in cryoscoop 384 to retainsample 340. Sample 340 in scoop 392 of cryoscoop 384 can be exposed tolaser warming by laser 390 after removal from coolant 356 to warm sample340 to desired temperature, e.g. room temperature or physiologictemperature. The warming of sample 340 can be performed to achievecritical warming rates (CWR) as further described herein.

The present description also includes a high-throughput warmingplatform. A high-throughput warming platform, for example, can includemultiple cryoscoops to process multiple samples. The cryoscoops can bereused once a sample has been warmed appropriately and transferred outof the warming platform into a receptacle in an appropriate environment.Many embodiments of a high-throughput warming platform can be used andall are within the scope of this description.

In FIG. 3B, light source 390 can be, for example, visible light or nearinfrared that is focused at a position with sample 340. As discussedherein, visible or near infrared light directed at the sample can causeheating of the sample. In the simplified block diagram of FIG. 3A, anoptional user input/output 334 a/334 b is provided. For example, thisinput can be a single button allowing an operator to initialize a test,or can be a more complex input such as a numerical keypad or of anumeric keypad allowing an operator to update parameters such asthreshold values used by station 300. In the configuration of FIG. 3A, acomputer identified as computing system 328 is used to perform thewarming. Computing system 328 couples to laser source 320 through I/Ocircuitry 324. I/O circuitry 324 can include, for example,digital-to-analog converters, analog-to-digital converters, switchableoutputs, etc.

FIG. 3C shows a schematic diagram of convective warming of sample 340.Cryoscoop 384 with sample 340 is placed in container 360 with embryomedia 362 and allowed to reach the desired temperature, e.g. room orphysiologic temperature. FIG. 3D shows a schematic diagram of laserwarming of sample 340. Cryoscoop 384 with sample 340 in scoop 392 isexposed to laser 390 to warm sample 340 to the desired temperature. FIG.3E shows photographs of sample 340 prior to warming, after laser warmingand after convective warming.

FIGS. 10A-10C show a schematic diagram of one exemplary embodiment of ahigh throughput laser warming station 1000. In FIG. 10A, warming station1000, e.g. Delta robot (IRB 360 FelxPicker®, ABB Group), is equippedwith scoop 1020 on handle 1006 to manipulate samples 1040 at high speed.In one exemplary embodiment, warming station 1000 may include pressurecontrol 1030 to apply a pressure or vacuum through handle 1006 (in thedirection of the arrow). Warming station 100 may also include base 1010to mount warming station 1000 to a laser chamber. Base 1010 may beconnected to pressure control 1030 through upper arms 1014 and lowerarms 1018. Upper arms 1014 and lower arms 1018 are flexibly connectedthrough rotatable attachments 1024. Scoop 1020 may be manipulatedrelative to a laser chamber mounted on base 1010. Base 1010 can move up,down, left, right, back and/or forward relative to scoop 1020 toretrieve and position sample 1040 at a desirable location relative to alaser chamber. In FIG. 10B, scoop 1020 and handle 1006 are connected topressure control device 1030 to pick up sample 1040. A vacuum may beapplied through handle 1006 by pressure control 1030 to provide inwardsuction or pressure in order to grab and hold sample 1040 in scoop 1020.In FIG. 10C, the process of high throughput laser warming and releaseinto culture media is shown. In FIG. 10C (i), scoop 1020 of warmingstation 1000 is manipulated to retrieve sample 1040 in cryogenic coolant1056 with a vacuum provided by pressure control 1030 to attach andretain sample 1040 in scoop 1020. Sample 1040 is removed from cryogeniccoolant 1056 and warmed by a laser in scoop 1020 as shown in FIG. 10C(ii). Sample 1040 is then transferred to culture media 1046 and releaseof the vacuum can release the warmed sample into culture media 1046 asshown in FIG. 10C (iii).

In FIGS. 13A-13D, one exemplary embodiment of scoop 1306 is shown in aperspective view, side view, top view and a bottom view, respectively.Scoop 1306 includes handle 1320 and posts 1310. A sample can be heldbetween posts 1310 in scoop 1306 and resting on base 1340. Base 1340 mayinclude an aperture therethrough for draining any residual liquid and inorder for scoop 1306 to retain a sample with minimal (or any)accompanying liquid. Posts 1310 can be protruding up from base 1340 totrap a sample within scoop 1306. Aperture in base 1340 can allow anycoolant or other liquid to pass through the aperture allowing a sampleto be held in scoop 1306. In other words, scoop 1306 acts as a sieve andholder when a sample is transferred from a liquid, e.g. liquid nitrogen,prior to warming the sample by a laser. The posts 1310 also holds thesample in position (e.g. a cryopreserved sample) when the cryoscoop islifted rapidly from liquid nitrogen for laser warming. Other embodimentsof scoop are possible and are within the scope of this description.

In one embodiment, a cooling platform, a sorting station and a warmingplatform may each be housed separately. In some embodiments, a coolingplatform, a sorting station and/or a warming platform may be housedtogether. In some embodiments, the cooling platform may be coupled to asorting station and/or a warming platform in sequence such that thecryopreserved sample may be transported to the sorting station such thatvitrified and crystallized samples can be separated. In someembodiments, the sorting station may be connected in sequence to awarming platform such that cryopreserved samples that are vitrifiedafter cryopreservation may be conveyed to enter the warming platform.Any combination of the cooling, sorting and warming components may behoused together and/or may be operably connected for processing thebiological samples.

A variety of biological samples can be cryopreserved according to thesystems and methods described herein. Biological samples can includehuman cells (e.g., pancreatic islet cells, HDF cells, stem cells, biopsysamples, etc.), mouse oocytes, zebrafish embryos, Xenopus laevisoocytes, coral larvae, or Lepidochelys olivacea embryos. In someembodiments, the sample can include germplasm—e.g., from a biopsy takenfrom a testis or an ovary from any animal or species. In otherembodiments, however, any tissue sample that can be loaded with acryoprotective agent and metal-containing laser absorber can be used inconnection with the systems described herein. Exemplary alternativesamples include, for example, neural cells, ganglia, stem cellspheroids, any biopsy from any soft tissue within the size parameterslisted in the immediately preceding paragraph. If the laser beam can bebroadened, additional exemplary samples include, for example, thecornea, skin, or other thin tissues. While described herein in thecontext of an exemplary embodiment in which the biological sample is azebrafish embryo, the systems and methods described herein can beapplied to a variety of materials.

The biological material can be any millimeter-sized biomaterial. In someembodiments, the term millimeter-sized sample can have a smallest lineardimension of less than about 5 mm. In one embodiment, the sample can beless than about 4 mm. In one embodiment, the sample can be less thanabout 3 mm. In one embodiment, the sample can be less than about 1 mm.In one embodiment, the sample can be less than about 0.5 mm In oneembodiment, the sample can be less than about 0.1 mm. In someembodiments, the biological sample can be between about 0.01 mm andabout 2.0 mm.

The biological material can be any microliter-sized biomaterial droplet.In some embodiments, the microliter-sized sample can have a volume ofless than 40.0 μl. In one embodiment, the microliter-sized sample canhave a volume of less than 10 μl. In one embodiment, themicroliter-sized sample can have a volume of less than 1 μl. In oneembodiment, the microliter-sized sample can have a volume of less than0.5 μl. In one embodiment, the microliter-sized sample can have a volumeof less than 0.1 μl. In some embodiments, the microliter-sized samplecan have a volume of between 0.1 μl and about 40.0 μl.

The biological samples may also include agents to promotecryopreservation. These agents can include, for example, cryoprotectiveagents and/or laser absorbers. Other agents that aid in thecryopreservation, sorting or warming processes may also be included inthe biological sample.

In one embodiment, a composition including a cryoprotective agent and alaser absorber may be microinjected into the biological sample. Thecryoprotective agent and/or the laser absorber may be in a medium thatis conducive to maintaining the integrity of the biological sample. Themedium, for example, can be a buffered medium or solution.

Also, while described herein in the context of an exemplary embodimentin which the cryoprotective agent includes propylene glycol, thecomposition, systems and methods described herein can involve the use ofany suitable cryoprotective agent. Exemplary suitable cryoprotectiveagents include, but are not limited to, combinations of alcohols,sugars, polymers, and ice blocking molecules that alter the phasediagram of water and allow a glass to be formed more easily (and/or athigher temperatures) while also reducing the likelihood of icenucleation and growth during cooling or thawing. In most cases,cryopreservative agents are not used alone, but in cocktails. In thecase of vitrification solutions, exemplary cryopreservative cocktailsare reviewed in Fahy et al., He, Xiaming, et al., Risco, Ramon, et al.and Choi, Jung Kyu, et al. and all incorporated herein by reference.(Fahy et al., Cryobiology 48(1):22-35, 2004; He, Xiaoming, et al.“Vitrification by ultra-fast cooling at a low concentration ofcryoprotectants in a quartz micro-capillary: a study using murineembryonic stem cells.” Cryobiology 56.3 (2008): 223-232; Risco, Ramon,et al. “Thermal performance of quartz capillaries for vitrification.”Cryobiology 55.3 (2007): 222-229; Choi, Jung Kyu, Haishui Huang, andXiaoming He. “Improved low-CPA vitrification of mouse oocytes usingquartz microcapillary.” Cryobiology 70.3 (2015): 269-272.) Additionalexemplary cryopreservative solutions can include one or more of thefollowing: dimethyl sulfoxide, glycerol, propylene glycol, ethyleneglycol, sucrose, trehalose, raffinose, polyvinylpyrrolidone, and/orother polymers (e.g., ice blockers and/or anti-freeze proteins).

In some embodiments, the cryoprotective agent may be present in thecomposition at various concentrations. In some embodiments, thecryoprotective agent may be present, for example, at a molarity of nomore the 6 M such as, for example, no more than 5 M, for example, nomore than 4 M, for example, no more than 3 M, for example, no more than2 M, for example, no more than 1 M, for example, for example, no morethan 900 mM, for example, no more than 800 mM, for example, no more than700 mM, for example, no more than 600 mM, for example, no more than 500mM, or for example, no more than 250 mM.

While described herein in the context of an exemplary embodiment inwhich the laser absorber is a gold nanorods, the technology describedherein can involve the use of a laser absorber of any suitable geometryand containing any suitable laser absorbing plasmonic material.Generally, the laser absorbing plasmonic material absorbs a narrow bandof laser energy in contrast to, for example, India Ink, which is a broadband absorber. Thus, in some embodiments, the laser absorber can includematerial effective at converting laser energy into heat such as, forexample, a metal such as gold, silver, titanium, and/or copper. In otherembodiments, the laser absorber can be an alternative plasmonic materialsuch as, for example, graphene. In some embodiments, the laser absorbercan include an additional material such as, for example, a silicon core.Thus, the laser absorber can include a plurality of materials generallyconstructed to include a core and a shell that at least partially coversthe core. The particular material or combination of materials used inthe laser absorber can be selected based, at least in part, on theparticular wavelength of the laser being used to warm the sample. Forexample, when using a diode laser with a wavelength of 800 nm, the laserabsorber can include a gold nanoshell with a silicon core. Similarly,the laser absorber can have any suitable geometry including, forexample, a rod shape, a sphere, a cube, a horn, a star, etc. Selectionof materials and geometry of the laser absorber can allow broad bandabsorption of various laser wavelengths from, for example, 200 nm to2000 nm. Thus, the laser can also be selected to excite at anywavelength suitable to match the absorption of the plasmonically activenanoparticle.

In some embodiments, the laser absorber is distributed throughout allcompartments of the cell. For a germinal cell, such a distributionincludes the presence of the laser absorber in the chorion and yolk. Fora somatic cell, such a distribution includes the presence of the laserabsorber in the cytoplasm and the nucleus.

In some embodiments, the laser absorbers are not within the cells but inthe exterior of the cells but in the fluid, e.g. media and/orcryoprotective agent, surrounding the cells. In these embodiments, thelaser absorbers can be included in the droplet that is cryopreserved andthus the cryopreserved sample includes the laser absorbers and allowsfor improved laser warming.

The system can incorporate droplet microfluidics to transport cellsthrough the system and/or add regents—e.g., a cryopreservation agent, anexcipient, culture medium, metal laser absorber (e.g., gold nanorods),etc.—to the cells prior to the cells being frozen. Multi-phasic dropletmicrofluidics may be used to introduce a plurality of components to thecells. In addition, once frozen, embryos could be stored and/ortransported to a laser warming jig that would quickly move embryos (oneat a time) under a laser pulse.

The present description can include methods for cryopreservation ofbiological samples. The methods may also include sorting and warmingbiological samples. FIG. 7 illustrates one exemplary embodiment of aschematic flow diagram for method 700 for cryopreservation of abiological sample. FIG. 7 is one embodiment and other methods orvariations of methods can also be used and are included in the scope ofthis description.

At step 710, the method can include preparing a sample forcryopreservation. The preparation can include, for example, treating thesample with agents that can assist in the cooling, sorting and/orwarming of the biological sample and preserving the integrity of thebiological sample during cooling, sorting and warming steps. In oneembodiment, the sample may be treated with cryoprotective agents and/orlaser absorbers as described herein. The sample, for example, may bymicroinjected with one or more cryoprotective agents and/or one or morelaser absorbers. The biological sample treated as described herein canbe bathed in a media that is amenable to maintaining the integrity ofthe biological sample.

Maintaining the integrity of the biological sample as used hereinrelates to preventing cellular membrane from disruption and release ofcellular contents due to loss of integrity of the membrane structure.Other forms of disruption of the biological sample that hinders recoveryof the biological activity, e.g. crystallization of sample, upon warmingafter cryopreservation should also be avoided. Other methods ofpreparing a biological sample may be used and are within the scope ofthis description.

The method can include step 720 of picking up the biological sample tobe cryopreserved. The sample can be picked up by a pick and print devicethat includes a syringe coupled to a pressure dispenser. The sample canbe picked up by the tip of a syringe. A pressure dispenser coupled tothe syringe can exert upward pressure from the tip of the syringe towardthe base of syringe. The tip of the syringe can be placed over thesample and the upward pressure of the syringe can lead to theassociation of the sample to the tip. The sample can remain associatedwith the tip as long as the upward pressure is maintained on thesyringe.

The syringe with the sample associated with the tip can be conveyed fromthe position where the sample is picked up toward a vessel at acryogenic temperature. The vessel, for example, can include cryogeniccoolant, e.g. liquid nitrogen. The syringe with the associated samplecan be moved to the desired position over the cryogenic environment. Thepressure dispenser can then be manipulated to exert downward pressurefrom the base of the syringe toward the tip to print or release thesample (step 730) into the cryogenic environment. Alternatively, thepressure dispenser can release the upward pressure from the tip to thebase of the syringe. The sample may be placed directly into thecryogenic coolant. Alternatively, the sample may be placed onto a highlyconductive material that is in a cryogenic environment, e.g. at acryogenic temperature. The highly conductive material can be a dish, aplate and the like. The highly conductive material may also be overlaidby a fibrous wicking material and the sample from the syringe tip isprinted or placed on the fibrous wicking material. Advantageously, thefibrous wicking material absorbs any moisture present or generated andmay reduce the incidence of crystallization with the sample. The printedsample may be placed on the highly conductive surface at a cryogenictemperature. The printed sample may also be allowed to roll or drop intothe cryogenic coolant from the highly conductive surface.

The cooling rates of the printed samples in the cryogenic environmentcan vary and are sufficient to achieve vitrification. In one embodiment,the cooling rate achieves at least the CCR for the sample. The coolingrates can be at least about 1,000° C./min. In some embodiments, thecooling rates can be between about 1,000° C./min and about 10,000°C./min. In some embodiments, the cooling rates can be about 10,000°C./min or faster.

The printed samples may, optionally, be transferred to a sorting stationas shown in step 740. The sorting device can also maintain a cryogenicenvironment as the sample is conveyed through the sorting device. Thechannels and the liquid in the sorting device can be functional atcryogenic temperatures. In other words, the channel walls and the liquidcan tolerate the cryogenic temperatures and the liquid is able totransport the samples at cryogenic temperature. The samples can passpast a light or laser source and a detector is able to determine thepresence of vitrified sample or a crystallized sample. Pressure can bereleased into the tubing upon detection of a crystallized sample leadingto the movement of the crystallized sample to a different path than avitrified sample as illustrated in FIG. 2B. Alternatively, the pressurecan be released into the channel upon detection of a vitrified sampleleading to the movement of the vitrified sample to a different path thana vitrified sample.

Sorting may also be conducted at other stages of the cryopreservationmethod. Sorting, for example, can be conducted as indicated in step740B. A sorting step can be conducted after the preparation step 710and/or after picking up of the sample, prior to printing the sample, todetermine the viability of the sample. Sorting at 740B can generally bedone without the sorting device at cryogenic temperatures since thesamples have not yet been cryopreserved.

After printing (step 750) or after sorting at (step 740), the samplesmay be stored at a cryogenic temperature for a desired length of time(step 750). The samples may be stored at cryogenic temperature for anylength of time, for example, a fraction of a second, a second, minute,an hour, a day, a month, a year or many years.

Methods of printing, storing and warming specimen may also be conductedwithout a sorting step and are within the scope of this description.

When a cryopreserved biological sample is needed for a desired purpose,the sample can be retrieved from storage and warmed (step 760). Avariety of methods can be used to warm the sample to the desiredtemperature, e.g. room temperature or physiological temperatures ornon-cryogenic temperatures. The sample can be removed from storage by,for example, a cryoscoop. The cryoscoop, for example, may include awell, or a scoop to hold the sample in place as shown, for example, inFIGS. 13A-13D. A laser source can be directed at the biological samplein the cryoscoop.

The cryopreservation methods described herein can lead to retention of ahigh percentage of the biological activity or cell viability of thebiological samples after warming from storage at a cryogenictemperature, e.g. below −80° C. Biologically active can refer to the useof the biological sample in a biological activity that would have beenperformed prior to cryopreservation. The biological activity of thecryopreserved sample is at least about 50 percent of the activityrelative to the activity of the sample prior to cryopreservation. Insome embodiments, the biological activity is at least about 60 percent,such as at least about 70 percent, at least about 80 percent, at leastabout 90 percent, at least about 95 percent of the activity relative tothe activity of the prior to cryopreservation.

The laser can be, for example, a near infrared (NIR) laser. The laser,for example, can provide a fluence rate from about 10⁶ W/m² to 10⁹ W/m².Other lasers and fluence rates may also be used and all are within thescope of this description.

The warming rates generated by the laser can vary and are fast enough tomaintain integrity of the sample. In one embodiment, the warming ratescan achieve the CWR. The warming rates can be at least about 300,000°C./min. In some embodiments, the warming rates can be between about400,000° C./min and about 24 million °C./min.

In some embodiments, this disclosure includes laser-assisted heating ofa composition or biological sample that includes a metal laser absorbersuch as, for example, gold nanorods. Gold nanorods are efficient forheating and to generate heat uniformly across a sample without toxicity.Thus, laser-assisted heating of a metal laser absorber can generate highheating rates uniformly inside a millimeter-sized biological sample andcan be used to rewarm any cryopreserved millimeter-sized biomaterialwhere the metal laser absorber can be disbursed. This technology can beexploited to allow a biological stock center (e.g., an aquaculturecenter, germplasm stock center, or a tissue bank) to store and shipvitrified biological samples. The recipient of the vitrified samples canthen rewarm the material using the technology described herein so thatthe material is suitable for, for example, research and/or commercialpurposes.

In one embodiment, the survival of zebrafish embryos postcryopreservation can be accomplished by an ultra-rapid nanowarmingapproach. This approach can include injecting zebrafish embryo with goldnanorods and low-concentration CPA and vitrified in liquid nitrogen. Thevitrified embryos are then warmed by laser heating of gold nanoparticlesachieving ultra-rapid rates of >10⁶° C./min. Using this approach,reproducible zebrafish embryo cryopreservation results can be attained.

In one exemplary embodiment, zebrafish embryos are microinjected withone or more cryoprotective agents, e.g. polypropylene glycol and/orlaser absorbers, e.g. gold nanorods. The gold nanorods and thecryoprotective agents are microinjected into a biological sample. Thesample is rapidly cooled to a temperature suitable for frozen storage.To rewarm the sample, the sample can be subjected to a NIR laser pulse.

A number of parameters can determine the optimal cryopreservation andwarming methods for a desired biological sample. The size of thespecimen and the size of the droplets can determine the optimalparameters for a sample. Warming of a cryopreserved sample may vary inthe strength of laser to be used and the concentration of the GNRsincluded in the sample prior to cryopreservation. For example, in someembodiments, a higher concentration of GNRs may be used incryopreservation and can be offset by the use of a lower strength oflaser during laser warming. As shown in FIG. 12B, at each concentrationof GNRs, there can be an amount of laser energy that can be applied tothe sample and maintain high cell viability. However, after a certainamount, increasing the amount of laser energy can quickly result inreducing the cell viability due to overheating (i.e., boiling thesample). In some embodiments, lower concentrations of GNRs can be usedand can be combined with higher laser energy while achieving uniformheating within the droplet and therefore higher cell viability comparedto when high concentrations of GNRs are applied. The size of the sample,the amount of desired laser energy to be applied to a sample, the amountof GNRs to be included can vary for each type of sample. An optimalmethod for cryopreservation and/or laser warming for a sample ofinterest may be determined by varying the parameters and identifying thedesired characteristics for sample of interest.

EXAMPLES Example 1 High Throughput Islets Cryopreservation Using a Pickand Print System

Islets were printed using the pick and print method with a pick andprint system. The system included 8 syringe tips and droplets wereprinted at a rate of 20 droplets per minute per tip. This resulted inthe system generating 160 droplets per minute. Islets were in 2Mpropylene glycol and 1M trehalose with GNR OD=1.

FIGS. 5A and 5B are photographs of the cryopreservation system of theembryo “pick & print” system. This embodiment includes a custom-built 3Dprinter, a pressure dispenser and a high precision tip. FIG. 5B is anoptical image of a printed droplet falling from the tip.

FIGS. 4A-4B are schematic diagrams of components of the cryopreservationsystem and photographs of the results of cryopreservation of droplet ofislets using the “pick and print” system. FIG. 4A is a schematic diagramof crystallization of islets in a CPA droplet (1.2 mm diameter, 2 Mpropylene glycol+1 M trehalose+3 OD GNP) when printed directly intoliquid nitrogen. FIG. 4B is a schematic diagram of the vitrification ofthe same CPA droplet as in FIG. 4A when printed onto cryogenic copperdish with a wicking paper as shown schematically in FIG. 4B. FIG. 4A andFIG. 4B include photographic images and XRD results at −170° C. ofsamples printed directly into liquid nitrogen and into a copper dishwith a wicking surface, respectively. FIGS. 4C-4E are photographicimages of 4 μl droplets with CPA and GNRs (1.2 mm diameter, 2 Mpropylene glycol±1 M trehalose+3 OD GNP). FIG. 4C is an image ofvitrified droplet without islets. FIG. 4D is a vitrified droplet withislets and FIG. 4E is a non-vitrified droplet with islets.

Example 2 Cryopreservation of Human Dermal Fibroblast (HDF) Cells UsingPick & Print and Laser Warming of the Cryopreserved Samples

High throughput droplet vitrification and laser warming for HDF cellswas performed. HDF cells in 4 μl droplets were cryopreserved usingpick-print technology to obtain vitrified samples. The droplets includedCPA of 2M propylene glycol and 1M trehalose and various concentrationsof GNRs. The vitrified cells were warmed with a laser. Laser warming wasperformed at a rate of 400,000 to 12,000,000° C./min. Cell viability ofthe HDF cells was tested after laser warming. HDF cell viability wastested with droplet volumes of 4 μl. Cell viability was examined atvarying laser energy and GNR concentrations (OD). Temperaturedistribution in droplets was also examined at various GNR OD.

FIGS. 11A-11C show the temperature distribution in droplets at variousGNR concentrations (OD). FIGS. 11A-11C show that as the concentration ofGNRs increases the amount of energy retained from the laser is greaterleading to an increase in the temperature of droplet especially inregions exposed to the laser (as opposed to the droplet face protectedby the cryoscoop). FIG. 12A shows the viability of the HDF cells atvarying laser energy and droplet volume. The GNR was fixed at GNR OD=4.6and laser pulse width=1 ms. The viability of HDF cells is affected bythe laser pulse energy. Bigger droplets (i.e., 4 μl) need more laserenergy compared with smaller droplets (i.e., 1 μl). In FIG. 12B, fixeddroplet volume is 4 μl and laser pulse width is 1 ms. Laser pulse energyand GNR concentration determined the cell viability. Lower GNRconcentration (i.e., OD=2.3) leads to higher viability than higher GNRconcentration (i.e., OD=4.6). In FIG. 12C, comparison of cell viabilityof control (i.e., untreated), CPA treated along (i.e., nocryopreservation), laser warming and convective warming groups. Dropletsize was 4 μl. Laser warming still had high cell viability compared toconvective warming.

Example 3 Preparation of Zebrafish Embryos for Cryopreservation andLaser Warming

Materials and Methods.

Animal Care and Culture. Wild-type zebrafish (Danio rerio) embryos wereobtained from the University of Minnesota Zebrafish Core. All care andwelfare for the animals met NIH animal care standards. Full details ofapproved protocols are listed with the Zebrafish Core-IACUC (protocol#1506-32642A). Zebrafish parent clutches and their embryos weremaintained at 28° C. under standard conditions as described inWesterfield.

Microinjection of Cryoprotectant and GNRs. The microinjection ofsolutions into zebrafish embryos has been well-established in theliterature. The high cell stage (t=3 h after fertilization) was chosento be a robust developmental stage for microinjection while stillallowing maximal uniformity of distribution of the injection throughoutthe embryo. The chorion was not removed in the experiments tomechanically protect the embryo during handling. The embryos wereinjected laterally through the chorion into the yolk. The volumes ofcryoprotectant and GNRs introduced were 9 nL in the yolk and 90 nL intothe chorionic space surrounding the embryo. Embryos (n=100 per group)were injected with solutions with N=1.2×10¹⁸ particles/m³ of GNRs, bothcoated with CTAB and PEG, and 0.2% WV India ink (Higgins Ink, model#HI44-011). Since India ink would plug up the injection needles, aslightly lower concentration was used than what was reported in theliterature. All the injected solutions were prepared in standard embryomedium (EM). Embryos for laser warming were microinjected with PG (2 M)and GNR-PEG (N=1.2×10¹⁸ particles/m³) using the same protocol asdescribed above. To test the efficacy of the injection and warmingprocesses, the experimental groups included (i) non-injected non-frozenembryos (n=383); (ii) microinjected (EM alone) nonfrozen embryos testedfor toxicity=200): (iii) microinjected (PG and GNRs) nonfrozen embryostested for toxicity (n=234); (iv) microinjected (PG and GNRs) embryos,frozen and “convectively warmed” (n=50); and (v) microinjected (PG andGNRs) embryos, frozen and “laser warmed” (n=223).

Estimation of SAR. To warm a zebrafish embryo from liquid nitrogentemperatures to room temperature (i.e., ΔT=221° C.) by a laser pulse(τ=1 ms), the required SAR can be estimated as SAR=ρCpΔT/τ=4.4×10¹¹W/m³, where ρ=990 kg/m³ and Cp=2 kJ/kg K are properties of ice. SinceSAR depends on GNR optical properties and laser fluence rate,comparisons were first made between theoretical predictions andexperimental results of GNR optical properties. First, the absorptioncross section, Cabs, was predicted from the DDA method as previouslyreported for GNRs and GNPs. Here the dipole density was assumed to be 4dipoles/nm, and the average of nine different laser GNR orientations(from 0° to 90°) were used to account for random GNR distribution insidethe embryo. Next, Cabs was experimentally measured using GNR warming ofa GNR solution with a cuvette heating method. This required measuringtemperature change resulting from laser warming (1064 nm CW laser.I1064SR0500B, Innovative Photonic Solutions) in a GNR solution(N=3.6×10¹⁸ particles/m³). In all the embryo warming cases, a similarconcentration of GNRs (N=1.2×10¹⁸ particles/m³) was injected, and theexperimentally determined absorption coefficient (μa=38.9 cm⁻¹) for theembryo was found. From this, the laser fluence rate needed to generatethe required SAR (i.e., 4.4×10¹¹ W/m³) was found to beI=SAR/μ_(a)=1.1×10⁸ W/m². From the laser fluence rate calibration, theoperating input conditions capable of generating the required fluencerate were found (see Laser Fluence Rate Calibration in SupportingInformation). The required laser fluence rate was verified by testingnumerous vitrified droplets of the same size as the embryo under thelaser and observing them melting and not refreezing. Very occasionally,refreezing was observed in droplets and embryos presumably due todifferential laser absorption between the Cryotop and the GNR-loadedsystem. In this case, the laser fluence rate was raised by the smallestpossible increment of 5 V to achieve melting without refreezing. Thiswas always achieved in ≤4 increments or ≤7.5% total change in voltagefrom our nominal 270 V setting. Higher reproducibility in GNR loading,Cryotop design, and laser conditions can reduce any possible refreezingeven further. The same laser conditions (I=1.2×10⁸ W/m², 1 ms pulsetime, N=1.2×10¹⁸ particles/m³) were consistently used in all thezebrafish embryo and HDF warming experiments to generate an adequateamount of heating (SAR).

GNR Distribution. To assess the nanoparticle distribution within theembryos, they were microinjected with fluorescent GNRs (L=110 nm, D=20nm) coated with Dylight 650 (emission peak at 670 nm) (nanoComposix, SanDiego, Calif., USA) and imaged with a fluorescence laser confocalmicroscope at 640 nm excitation (Nikon C2si, NY, USA). After the initialconvection from the microinjection, the GNRs continued to move bypassive diffusion for 3-4 h prior to imaging.

Embryo Survival Analysis. Twelve hundred embryos were examined atdifferent time points after microinjection for survival. The selectedtime points were 1 h, 3 h, 24 h, and 5 days after microinjection (fortoxicity tests). For the first three time points, embryos wereconsidered alive if they were developing and moving within the chorionbetween consecutive time points. At the day 5 time point, an embryo wasconsidered normal in development if it had hatched and was able to swimupright in the water column, had proper cardiac development, eye andtail musculature development, fins, and a functional swim bladder. Anyfish that did not match these criteria was considered abnormal in theirdevelopment and a failed survival.

Cooling and Laser Warming Experiment. Recent studies measured coolingrates of 69 000° C./min or higher for a liquid nitrogen cooling 0.1 μldroplet on a Cryotop (Kitazato Corp., Fuji, Japan). Since the criticalcooling rate for vitrification of 2 M PG is 50 000° C./min and theCryotop can exceed it, the Cryotop was chosen for this study. As thecommercially available Cryotop is only 0.4 mm wide, it was not wideenough for a zebrafish embryo (D>1 mm, considering the chorion), andmodifications were made to extend the width of the polypropylene bladeto 1.5 mm. Simulated cooling rates with this modified design suggestrates more than 50 000° C./min are possible, and this approach was usedin all subsequent embryo cooling.

To move embryos for vitrification rapidly into the liquid nitrogen andquickly bring them from the liquid nitrogen under the laser beam, a pickand print system with a sorting station and a warming platform with acryoscoop as described herein can be used. The embryos can be pick andprinted into a copper dish with a wicking surface in liquid nitrogen.The embryo was held in liquid nitrogen until successfully cryopreservedat liquid nitrogen temperatures and analyzed for vitrification. In rarecases, embryos would turn white or opaque, suggesting ice crystalformation during cooling, and these were discarded. After equilibration,the embryos were quickly and reproducibly moved to a position of focusunder the laser beam such that warming could be achieved in one laserpulse. The movement was initiated only after the optimal laserparameters had been validated such as voltage, pulse time, and spot sizeto achieve the fluence rate to produce the warming rates needed. FIG. 8is a plot of Laser fluence rate calibration for 1064 nm laser. Fluencerates were calculated by using a power meter to measure energy per pulsefor different voltage and pulse width conditions. The spot size fixed tothe maximum of 2 mm. The average value of 3 trials is reported here(standard deviation was less than 0.1% for all points).

FIG. 9 is a plot showing the minimum laser fluence rate required to melta single 1 ul droplet containing 2M propylene glycol and 1M Trehalosebut varying concentrations GNR. The attenuation coefficient isproportional to the number of GNR in the droplet. It also shows thatamount of laser power that would be absorbed by the droplet base onBeers law.

Specifically, one laser pulse at 270 V, 1 ms pulse time, and 2 mm spotsize were used to warm embryos. The laser fluence rate provided by thelaser was determined characterization of Laser Fluence Rate to beapproximately 1.1×108 W/m2.

After the laser warming, the cryoscoop can be removed and the embryo canbe placed into embryo medium for further analysis. For the convectivewarming case (n=50), microinjected embryos were cooled as before withthe modified Cryotop into liquid nitrogen. However, after equilibration,the embryos were immersed into embryo medium at 25° C. for warming. Theexperimental and control groups were then cultured at 28° C. andmonitored regularly up to day 5.

Additional details for methodology related to cryopreservation and laserwarming are in Khosla et al. “Gold Nanorod Induced Warming of Embryosfrom the Cryogenic State Enhances Viability” ACSNANO July 2017,incorporated herein by reference in its entirety including thesupplementary information and Khosla et al, “Characterization of LaserGold Nanowarming: A Platform for Millimeter-Scale CryopreservationLangmuir 2019, 35, 23, 7364-7375, also incorporated herein by reference.

Although specific embodiments have been illustrated and describedherein, any arrangement that achieve the same purpose, structure, orfunction may be substituted for the specific embodiments shown. Thisapplication is intended to cover any adaptations or variations of theexample embodiments of the invention described herein. These and otherembodiments are within the scope of the following claims and theirequivalents.

What is claimed is:
 1. A cryopreservation system comprising: a coolingplatform comprising a syringe holder, the syringe holder comprising oneor more syringes, each syringe with a tip configured to pick and print abiological sample, the syringe coupled to a pressure dispenser whereinthe tip of the syringe picks up the biological sample when the pressuredispenser exerts upward pressure from the tip toward the base of thesyringe and prints the biological sample into a cryogenic environmentwhen the pressure dispenser exerts downward pressure toward the tipand/or releases the upward pressure toward the base of the syringe. 2.The system of claim 1 wherein the syringe is movably engaged within thesyringe holder to move from a pick position to a print position.
 3. Thesystem of claim 1 wherein the biological sample is selected from asingle cell, multiple cells, aggregates of cells, germplasm, embryos oroocytes.
 4. The system of claim 1 wherein the biological sample is atleast 0.01 mm in diameter.
 5. The system of claim 1 wherein thebiological sample is a droplet between about 0.1 μl and about 40.0 μl.6. The system of claim 1 wherein the biological sample comprises laserabsorbers and/or cryoprotective agents.
 7. The system of claim 1 whereinthe cooling platform is a high throughput system comprising two or moresyringes with tips for picking and printing multiple biological samples.8. The system of claim 1 further comprising a sorting station, whereinthe sorting station comprises a sorting device with channels sized forflow of the biological samples in a fluid, a light source, a detectorand a pressurized air tank operably connected to the detector and abuffer reservoir, wherein the detector can detect a vitrified samplefrom a unvitrified sample and the pressurized air tank operablyconnected to send a pulse of pressurized air to the buffer reservoirthrough an airline resulting in a pulse of buffer fluid entering thechannel in the sorting station, wherein the sorting station is amicrofluidics based sorting station and wherein the sorting stationsorts the biological sample at a cryogenic temperature.
 9. The system ofclaim 1 further comprising a warming platform, the warming platformcomprising a cryoscoop for removing the biological sample from acryogenic environment, wherein the warming platform further comprises alaser for warming the biological sample from a cryogenic temperature toa desired temperature.
 10. A method for cryopreservation of a biologicalsample comprising: picking up the biological sample with a syringehaving a tip, wherein the syringe is engaged in a syringe holder of acooling platform, the syringe coupled to a pressure dispenser whereinthe tip picks up the biological sample when the pressure dispenserexerts upward pressure from the tip toward the base of the syringe; andprinting the biological sample wherein the sample is printed when thepressure dispenser exerts downward pressure toward the tip and/orreleases the upward pressure toward the base of the syringe, wherein thebiological sample is printed into a cryogenic environment.
 11. Themethod of claim 10 wherein the cooling platform is a high-throughputsystem comprising one or more syringes.
 12. The method of claim 10wherein about 20-400 biological samples are cryopreserved in 1 minute.13. The method of claim 10 wherein the biological sample is printed ontoa fibrous wicking material resting on the surface of a highly conductivematerial and wherein the highly conductive material and the fibrouswicking material are resting in a cryogenic coolant.
 14. The method ofclaim 10 wherein the biological sample is selected from a single cell,multiple cells, aggregates of cells, embryos or oocytes.
 15. The methodof claim 10 wherein the biological sample is a droplet between about 0.1μl and about 40.0 μl.
 16. The method of claim 10 further comprisingsorting the biological sample, wherein the sorting comprises separatingvitrified biological samples from crystallized biological samples,wherein pressure is applied when vitrified or crystallized samples aredetected by a detector to separate the vitrified biological samples fromthe crystallized biological samples.
 17. The method of claim 16 whereinthe sorting is performed with a microfluidic sorting system.
 18. Themethod of claim 10 further comprising a method for warming thecryopreserved biological sample with a warming platform, wherein thewarming method comprises removing the biological sample from a cryogenicenvironment by a cryoscoop and warming the biological sample with laserassisted warming.
 19. A sorting station comprising a sorting device withchannels sized for flow of biological samples in a fluid, a lightsource, a detector and a pressurized air tank operably connected to thedetector and a buffer reservoir, wherein the detector can detect avitrified sample from a unvitrified sample and the pressurized air tankoperably connected to the detector to send a pulse of pressurized air tothe buffer reservoir through an airline resulting in a pulse of bufferfluid entering the channel in the sorting station to alter the pathwayof the biological sample in the channel closest to the distal end of thepulse of the buffer fluid.
 20. The sorting station of claim 19 whereinthe sorting station separates the vitrified samples and the unvitrifiedsamples into different channels.